In recent years, as a result of concerns related to energy supplies for transportation, as well as other environmental problems such as global warming and aerial pollution, the development of alternative energy sources to oil has become an extremely important issue. Plant biomass is the most plentiful renewable energy source on earth, and holds great promise as an alternative energy source to oil. The main component of plant biomass (lignocellulose) is composed of polysaccharides such as celluloses and hemicelluloses (including xylan, arabinan and mannan), as well as lignin and pectin and the like. These polysaccharides are hydrolyzed by a large variety of glycoside hydrolases to form monosaccharides such as glucose and xylose, which can then be used as biofuels or the raw materials for chemical products.
Lignocellulose is recalcitrant due to its highly complex structure, and is difficult to degrade or hydrolyze with a single cellulolytic enzyme. Accordingly, among the various polysaccharides, hydrolysis of cellulose generally requires three types of glycoside hydrolase enzymes, namely an endoglucanase (endo-1,4-β-D-glucanase, EC 3.2.1.4), an exo-type cellobiohydrolase (1,4-β-cellobiosidase or cellobiohydrolase, EC 3.2.1.91, EC 3.2.1.176), and a β-glucosidase (EC 3.2.1.21). On the other hand, hemicelluloses include xylan, arabinan, and mannan and the like, and the structure varies depending on the plant. For example, in the case of hardwoods and herbaceous plants, xylan is the main structural component. Hydrolysis of xylan requires a xylanase (endo-1,4-β-xylanase, EC 3.2.1.8) and a β-xylosidase (3.2.1.37).
In conventional bioethanol production using lignocellulose as a starting resource, hydrolysis processes using high solid loading (30 to 60% solid loading) have been tested with the aim of achieving a more energy-efficient conversion to ethanol. However, in this type of lignocellulose enzymatic hydrolysis using high solid loading, the viscosity of the biomass slurry is high, and the hydrolysis reaction of the lignocellulose tends to proceed poorly. Accordingly, by using a thermostable enzyme and performing the enzymatic hydrolysis treatment at a high temperature of 80° C. or higher, the rate of the hydrolysis reaction can be increased, and the viscosity of the biomass slurry can be reduced, which is expected to enable a shortening of the hydrolysis reaction time and a reduction in the amount of enzyme required. As a result, for all of the various glycoside hydrolases, the development of enzymes having superior thermostability is very desirable.
Many thermostable glycoside hydrolases have been obtained by isolating and identifying thermophilic microorganisms that exist in high-temperature environments, cloning genes from these isolated and cultured microorganisms, determining the DNA sequence, and then expressing the DNA using E. coli or filamentous fungi or the like. Particularly in the case of xylanases required for the hydrolysis of the hemicellulose xylan, large numbers have already been isolated from thermophiles, filamentous fungi, and Archaea and the like for purposes such as lignocellulose hydrolysis and pulp processes and the like. For example, Patent Document 1 discloses a xylanase derived from Acidothermus cellulolyticus, the xylanase exhibiting enzymatic activity at 60 to 80° C. Patent Document 2 discloses a xylanase derived from an anaerobic thermostable bacterium isolated from a New Zealand hot spring, and this xylanase also exhibits enzymatic activity at 60 to 80° C. Patent Document 3 discloses a xylanase derived from a Bacillus bacterium isolated from the soil, the xylanase having an optimum temperature of 80° C. Non-Patent Document 4 reports a xylanase derived from Acremonium cellulolyticus, the xylanase having an optimum temperature in the vicinity of 60 to 80° C. Tests aimed at further improving the thermostability have also been conducted, and Patent Documents 4 to 7 disclose xylanases for which the thermostability has been improved by substituting amino acids of natural enzymes. Patent Document 8 discloses that by truncating a natural xylanase having an enzymatically active domain and a carbohydrate binding module connected by a linker, by removing either the carbohydrate binding module or the carbohydrate binding module and the linker, production of the enzyme within the host could be increased. Almost all of the above enzymes have optimum temperatures of 60 to 80° C., and further improvements in the thermostability are still required.
On the other hand, Patent Documents 9 and 10 and Non-Patent Documents 1 to 3 disclose examples of hyper-thermostable xylanases isolated from specific bacteria and filamentous fungi, and xylanases having an optimum temperature exceeding 85° C. have been reported. Patent Document 9 reports a xylanase with an optimum temperature of 90° C. derived from Rhodothermus marinus, but the specific activity at 90° C. is only about 26 nkat/mg protein (=about 1.6 U/mg protein), which is very low.